Cell lines are used in basic research as well as in applied disciplines like protein production, drug discovery and toxicity testing and are also used for therapy in regenerative medicine approaches. The problem of existing cellular models is that they are either lacking biological relevance or the cells are not available in sufficient amounts. However, the elucidation of molecular processes heavily depends on suitable cell lines. Therefore, cells reflecting in vivo properties which can be produced in sufficient amounts are of high interest for modern life sciences.
Basically, cells can be derived from two different sources: they can be isolated as primary cells from individuals/animals, or they are provided by cell lines. The advantage of primary cells is that they closely reflect the properties of cells in vivo. This high biological relevance is unfortunately linked with several drawbacks like tedious and time consuming isolation procedures, a high batch to batch variability and complex cultivation conditions. The major limitation which hinders a more widespread use of primary cells is their limited proliferation capacity which significantly restricts expansion. Cell lines, on the other hand, are either isolated from tumours or generated upon spontaneous or induced immortalisation of primary cells. Cells of cell lines are unlimitedly available, homogeneous, show constant properties, are easy to handle and to maintain. However, so these cells lack many features and markers of tissue they were isolated from.
A major limitation of the establishment of new cell lines is (i) the unpredictability e.g. for spontaneous immortalisations of primary cell material, (ii) the fact that conventional immortalisation regimens solely with the help of known immortalising genes like the SV40 large T antigen (TAg) or viral oncogenes of the papilloma virus E6/E7 lead to the drastic alteration of the cell physiology. Therefore these cell lines do no longer reflect the physiology of the in vivo state and therefore the biological relevance is missing in these cell systems (iii) no system exists that is universal, meaning there is no single immortalising gene that is capable of establishing cell lines from any cell type, from different donors, and from different species.
Spontaneous immortalisation can occur from primary cell material derived from a tumour or in rare cases also from non-malignant or benign primary cells. These cell lines are easily maintained and expanded until a robustly proliferating cell line is established. The overall success rate of this process is low and therefore huge amounts of primary cell material have to be available. Due to the random nature of this process the properties of the resulting cell lines cannot be influenced and the resulting cell line mostly does not appropriately reflect its origin.
Conventional immortalisation regimens usually employ the recombinant expression of oncogenes like e.g. the catalytic subunit of the human telomerase (hTert), SV40 large T antigen (TAg), the polycomb protein Bmi1 or viral oncogenes E6/E7 from the human papilloma virus, the viral oncogenes E1A/E1B. A frequently used immortalisation gene is the hTert, which has proven useful for the expansion of a wide variety of cell types (Bodnar A G, Ouellette M, Frolkis M, et al. Science. Jan. 16, 1998; 279 (5349):349-352.). It acts by maintaining the ends of the telomeres. Telomeres are stretches of repetitive DNA at the very end of the linear chromosomes. These stretches cannot be replicated by DNA polymerases during replication and therefore the telomeres progressively shorten with every replication round. This “end-replication problem” can be overcome by the recombinant expression of hTert which eventually can lead to immortalisation. Cell types expanded and used for tissue engineering include e.g. bovine adrenocortical cells (reference), human dermal endothelial cells (Yang J et al., Nat Biotechnol. March 2001; 19(3):219-224) and human mesenchymal stem cells (Simonsen J L et al., Nat Biotechnol. June 2002; 20(6):592-596).
However, prolonged constitutive expression of telomerase induces changes in gene expression that lead to a premalignant phenotype (Milyaysky M, et al., Cancer Res. Nov. 1, 2003; 63(21):7147-7157). In addition, the use of hTert is restricted to certain human cell types as others need the concerted action of several genes for efficient immortalisation (Kiyono T et al., Nature. Nov. 5, 1998; 396(6706):84-88). Furthermore, human cells require different immortalisation strategies to cells from other mammals like e.g. murine cells (Rangarajan, A., et al., Cancer Cell, August; 6 (2):171-83, 2004). This leads to the fact that hTert fails in the establishment of murine cell line. Another gene that has been frequently used for immortalisation is TAg. TAg is a viral oncogene that is known to modulate the activity of a number of proteins. Among those, p53 and pRb are regarded as the most important ones for immortalisation. Binding of TAg to p53 inhibits p53 mediated growth control. However, the inactivation of p53 also leads to the interference with the DNA damage response which in turn leads to DNA damage of host cell chromosomes. TAg also interferes with the pRb (retinoblastoma protein) tumour suppressor. Through this interaction/inhibition the transcription factor E2F is activated, which is responsible for the progression of the cell through the cell cycle. However, TAg can only be employed for the establishment of rodent cell lines. For the immortalisation of human cells (i) TAg alone is not sufficient and therefore a second oncogene has to be used which is usually hTert and (ii) the resulting cell lines are often characterized by a grossly altered karyotype which is most probably due to the inactivation of the p53-driven DNA damage response.
Other genes that facilitate immortalization are the E6 and E7 proteins from the human papillomavirus. They interfere with cell cycle control and the regulation of apoptosis. E7 inhibits by binding to the pRB family members their function and thereby facilitates cell cycle progression as the cells enter the S phase by disrupting pRb-E2F complexes. E6 on the other hand is known to promote the degradation of p53 and thereby to disrupt the growth control by p53. Another function of E6 is the induction of telomerase activity which supports the immortalization of cells by maintaining telomere length. For the immortalisation of human cells both proteins—E6 and E7—are required. However, the combination of these genes works mainly for the immortalisation of epithelial cells. Another drawback is that these genes induce genomic instability so that the established cell lines are polyploid.
Other oncogenes seem to be cell type specific as they only work for a very limited number of cell types of certain species, for examples HoxA9/HoxB9 for murine macrophages (Wang et al., Nat Methods. 2006 April; 3(4):287-93.). Other examples are (i) the v-myc oncogene which allows the immortalization of murine/rodent macrophages (Pirami et al., Proc Natl Acad Sci U.S.A. 1991 Sep. 1; 88(17):7543-7.), (ii) the Epstein Barr Virus which readily immortalizes human B lymphocytes (Henle et al., Science. 1967 Sep. 1; 157(792):1064-5.) or (iii) the Polyoma middle T antigen which establishes murine embryonic endothelial cells (Williams et al., Cell. 1989 Jun. 16; 57(6):1053-63.)
In summary such conventional immortalization techniques very often lead to drastically altered or mutated cell lines. To circumvent this issue approaches were undertaken in which the effects of the immortalization genes are controllable. For example in WO 2010/000491 A1 at least two immortalizing genes are put under the control of a transcriptional regulation. In this setting the immortalizing genes are introduced into the primary cells and activated through an external stimulus which leads to the immortalization of the respective primary cells. The withdrawal of the external stimulus leads in turn to the inactivation of the immortalizing genes. This step efficiently induces in these immortalized cell lines a senescent phenotype—a cellular state which is characterized by a complete growth arrest and which is a tumor suppressor mechanism. Therefore the technology described in WO 2010/000491 A1 generates cell lines which are only useful for a very specialized field of research.
Thus, there is still a need in the art for a species and cell-type independent method for producing cell lines from a variety of primary cells.